Ferguson sandbox 1

Isocitrate dehydrogenase (IDH) is an enzyme that is used during the third step of the citric acid cycle. This biological reaction is an essential process that is used to create molecules that are used for cellular energy. In this step it catalyzes the oxidative decarboxylation of isocitrate meaning that CO2 is released from the isocitrate. In addition coenzyme NAD+ is converted to an NADH. This reaction results in an alpha-ketoglutarate molecule which is then moved on to the fourth step of the citric acid cycle. Along with its catabolic role in the Krebs Cycle, IDH has been shown to play an important role in cellular defense against oxidative damage as a source of NADPH.

Structural Analysis
Isocitrate dehydrogenase is SCOP classified as an alpha beta structure. Its secondary composition consists of mainly alpha helices and beta sheets which are arranged into three layer alpha beta alpha sandwich structures. . The entire protein consists of two side by side sandwich structures that face opposite directions and form a homodimer. This then causes the protein's two active sites to face opposite directions. These two groups make up the A and B subunits of isocitrate dehydrogenase. Subunit A has a more well defined electron density, while subunit B has a more disordered and discontinuous electron density. These two subunits also adopt different conformations. Alpha helix 10 of the small domain is completely unwound and intrudes into the active site in subunit A; in subunit B it is partially unwound and is extended into subunit A. Alpha helix 9 in both subunits is expanded outwards. In human isocitrate dehydrogenase there are 4 subunits. HcIDH, like other forms of IDH, consists of three domains: a large domain, a small domain, and a clasp domain. The large domain has a typical Rossman fold, the small domain forms an alpha/beta sandiwch structure, and the clasp domain folds as two two-stranded anti-parallel beta sheets stacked on top of each other.

The Active Site
The active site of Isocitrate dehydrogenase binds the NAD+ or an NADP+ molecule as well as an Mg2+ or Ca2+(Active Site ). This metal ion seems to be essential for catalysis. Side chains of Asp 279 form hydrogen bonds with Ser94 (Human isocitrate dehydrogenase). Isocitrate is able to bind to the active site using about 8 amino acids such as tyrosine, serine, asparagine, arginine, arginine, arginine, tyrosine, and lysine. Across species these are not perfectly conserved but they are replaced with residues of similar properties and also are found in similar areas of the protein. Below is a picture of Porcine active site with all of its residuals and ligands.

In the structure of HcIDH quaternary complex, an isocitrate molecule and a Ca2+ ion are found to bind to the active site of each subunit. The isocitrate substrate interacts with Thr77, Ser94, Arg100, Arg109, Arg132, Tyr139, and Asp275 of one subunit; Lys212, Thr214, and Asp252 of the adjacent subunit; 3 water molecules; NADP; and Ca2+.



The blue is the Mg+ ion next to the ligand which is surrounded by the residues of that active site.

Mechanism
The mechanism described is for porcine isocitrate dehydrogenase because is is better understood than the human mechanism. The Citrate Molecule is converted to alpha-ketoglutarate. In the first step the alcohol group off the alpha-carbon is deprotonated by the Tyr residue. The electrons move to the oxygen atom to form a double bond (ketone). The remaining alpha carbon hydrogen is removed using NAD+/NADP+ as an electron accepting cofactor. A carboxyl group pushes electrons down so an oxygen steals a nearby proton off a Lysine amino acid. The Tyr deprotanates the carboxylic acid resulting in electron pushing that ejects a CO2. The two negatively charged oxygens on the opposite side of the molecule are stabilized by the Mn2+. The double bond that was formed between the alpha and beta carbon removes a proton from the Tyr residue and the oxygen returns the a keytone and the alpha-ketoglutarate is formed. This is illustrated in the figure below.

The mechanism by which isocitrate is converted to alpha-ketoglutarate

Regulation
In Escherichia coli and other bacteria the IDH is regulated by the reversible phosphorylation of Ser113 at the active site which is catalyzed by IDH kinase/phosphatase (IDHK/P). The images at the left and at the right above correspond to one representative Isocitrate dehydrogenase, i.e. the crystal structure of Isocitrate dehydrogenase from Escherichia coli (1sjs). In mammalian IDH, the regulation of the enzyme is still unclear. Mammalian and bacterial IDH have less than 20% sequence identity but the mammalian structure is very similar to the structure found in bacteria. Sequence alignments indicate mammalian IDH has a strictly conserved serine at a position equivalent to Ser113 in E.coli. These results suggest that, like bacterial IDH, mammalian IDH may contain a phosphorylation site and may be regulated by kinases and phosphatases as well.

Inhibition


This step an irreversible reaction. It must be carefully regulated to avoid depletion of isocitrate in the body. The reaction is started by substrate availability which are: isocitrate, Mg2+, NAD+ or NADP+. If these substrates are not present the process will not carry forward. The reaction is inhibited by the removal of NADH from the presence of the citric acid cycle. The product is also inhibited by ATP feedback. This feedback inhibition is a competitive inhibitor. Since the citric acid cycle is used to produce energy molecules (ATP), an abundance of product will shut down the cycle.

Involvement in Cancer
Mutations in Isocitrate Dehdryogenase isotypes, IDH1 and IDH2 have recently been discovered in CNS cancers like Gliomas, and a number of types of leukemia, including Acute Myeloid Leukemia. This discovery has been extended to prostate cancer, colon cancer, and thyroid cancer as well since 2009. Mutations to residue Arg 132 are the most common cancer inducing mutation. This residue directly binds the citrate. Instead of producing alpha-ketoglutarate, this mutant produces 2-hydroxyglutarate.

There are two competing theories as to how this mutation to IDH is cancer inducing. The first theory stipulates that the 2-hydroxyglutarate (2HG) produced inhibits proline hydroxylase (PHD). PHD is responsible for telling the cell if there is ample oxygen present for oxydative phosphorylation by hydroxylating Hypoxia Inducing Factor (HIF). When 2HG inhibits PHD, HIF is transported to the nucleus where it is believed to abberantly activate gene transcription, leading to cancerous conditions.

The alternative theory, which has recently been gainging significant support, states that IDHs hypermethylate DNA sites which code for proteins assoicated with cell differentiation, repressing these genes. Mutant IDHs however produce 2HG which is a competitive inhibitor of TET2, a protein which hydroxylates methylated cytosines, effectively resulting in demthylation. With TET2 inhibited, the cell differentiation inducing genes remain inactive resulting in undifferentiated cancer cells.

Additional Resources
For Additional Information, See: Citric Acid Cycle For Additional Information, See: Cancer

Isocitrate dehydrogenase
3blx – hIDH - yeast

2uxq – DpIDH – Desulfotalea psychrophila

3dms – IDH – Burkholderia pseudomallei

2e0c, 2dht - StIDH – Sulfolobus tokodaii

2iv0 – IDH – Archaeoglobus fulgidus

1zor – IDH – Thermotoga maritima

2b0t – IDH – Corynebacterium glutamicum

1xgv, 1v94 - ApIDH – Aeropyrum pernix

1pb3, 1sjs, 6icd, 7icd, 3icd – EcIDH – Escherichia coli

1idd, 1idf, 4icd - EcIDH (mutant)

IDH+citrate
2qfw – yIDH+isocitrate

3blv – yIDH+citrate

2uxr – DpIDH+isocitric acid

1itw – AvIDH+isocitrate+Mn – Azotobacter vinelandii

1lwd – IDH+isocitrate+Mn - pig

1pb1, 1p8f – EcIDH+isocitrate

1cw7, 5icd, 8icd - EcIDH +isocitrate+Mg

1cw1 - EcIDH (mutant)+isocitrate+Mn

1gro, 1grp - EcIDH (mutant)+isocitrate+Mg

1hqs – IDH+citrate – Bacillus subtilis

IDH+NADP
1t09 – hIDH+NADP - human

2qfv – yIDH+NADP

2e5m – StIDH+NADP

2cmj – mIDH+NADP – mouse

1tyo - ApIDH+NADP

1j1w – AvIDH+NADP – Azotobacter vinelandii

9icd - EcIDH +NADP

1iso - EcIDH (mutant)+NAD

IDH+α-ketoglutarate
2qfy - yIDH+α-ketoglutarate

1cw4 – EcIDH (mutant)+α-ketoglutarate

1ika - EcIDH + α-ketoglutarate+Ca

IDH ternary complexes
3inm – hIDH (mutant)+NADPH+α-ketoglutarate+Ca – human

1t0l – hIDH+NADP+isocitrate+Ca

2qfx - yIDH+NADPH+α-ketoglutarate+Ca

3blw – yIDH+citrate+AMP

2cmv - mIDH+NADP+citrate

2d4v – IDH+NAD+citrate – Acidithiobacillus thiooxidans

2d1c – IDH+NADP+citrate – Thermus thermophilus

1xkd – ApIDH+NADP+citrate

1hj6 – EcIDH (mutant)+NADP+isopropylmalate+Mg

1idc - EcIDH (mutant)+oxalosuccinate+Mg

1ai3 - EcIDH +isocitrate+Mg+NDP

1ai2 - EcIDH +isocitrate+NADP+Ca

1ide - EcIDH (mutant)+isocitrate+Mg+NADP

1bl5 - EcIDH +α-ketoglutarate+NADP+Mg